en
Scientific article
Open access
English

Reversible immortalization of human primary cells by lentivector-mediated transfer of specific genes

Published inMolecular therapy, vol. 2, no. 4, p. 404-414
Publication date2000
Abstract

We exploited the ability of lentiviral vectors to govern the stable transduction of cells irrespective of their cycling status to induce the reversible immortalization of human primary cells. First, bicistronic HIV-derived lentiviral vectors expressing GFP- and the HSV1 thymidine kinase and containing the LoxP sequence in their LTR (HLox) were used to transduce HeLa cells. Cre expression led to efficient proviral deletion, and unexcised cells could be eliminated by ganciclovir treatment. A human liver biopsy was then exposed to a combination of HLox vectors that harbored either the SV40 large T (TAg) or the human telomerase (hTERT) DNAs in place of GFP. This led to the isolation of liver sinusoidal endothelial cell (LSEC) clones that exhibited an immortalized phenotype while retaining most of the features of primary hLSEC. Complete growth arrest of these cells was observed in 2 days of Cre expression, and the resulting stationary culture could be kept for at least 2 weeks. Transduction of human adult pancreatic islets with HLox vectors coding for Tag and Bmi-1 also induced the proliferation of insulin-positive cells. These results indicate that lentivectors can be used to mediate the reversible immortalization of primary nondividing cells and should allow for the production of large supplies of a wide variety of human cells for both therapeutic and research purposes.

Keywords
  • Animals
  • Antigens, Polyomavirus Transforming/genetics/metabolism
  • Biopsy
  • Blotting, Western
  • Cell Differentiation
  • Cell Division
  • Cell Transformation, Viral
  • Cell Transplantation
  • DNA Primers/chemistry
  • Endothelium, Vascular/cytology/metabolism
  • Flow Cytometry
  • Gene Transfer Techniques
  • Genetic Therapy/methods
  • Genetic Vectors
  • HeLa Cells/cytology/metabolism
  • Humans
  • Immunohistochemistry
  • Integrases/metabolism
  • Islets of Langerhans/cytology/metabolism/virology
  • Lentivirus/genetics
  • Liver/cytology/metabolism
  • Mice
  • Mice, Nude
  • Nuclear Proteins/biosynthesis/metabolism
  • Polymerase Chain Reaction
  • Recombination, Genetic
  • Telomerase/genetics/metabolism
Citation (ISO format)
SALMON, Patrick et al. Reversible immortalization of human primary cells by lentivector-mediated transfer of specific genes. In: Molecular therapy, 2000, vol. 2, n° 4, p. 404–414. doi: 10.1006/mthe.2000.0141
Main files (1)
Article (Published version)
accessLevelPublic
Identifiers
ISSN of the journal1525-0016
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206downloads

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